RNA Isolation and Northern Blot Analysis

Total RNA was isolated from mouse tissues using RNA STAT-60 reagent (Tel-Test) following the manufacturer’s instruction [11 (link)]. Fifteen micograms of total RNA was denatured and electrophoresed on 1% agarose-formaldehyde gels. Uniformity of sample loading was verified by UV visualization of ethidiumbromide-stained gels before transfer to Nylon membranes. The cDNA probes for PAMM were amplified by PCR using a cDNA clone from A.T.C.C. as templates. ³²P-labelled cDNA was prepared using the random priming method (Invitrogen). Hybridization was performed using QuckHyb buffer (Strategene) at 65°C for 2 h or overnight. Then membranes were washed once with 2 × SSC and once with 0.1 × SSC, 1% SDS for 20 min at 65°C.

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Guo F., He H., Fu Z.C., Huang S., Chen T., Papasian C.J., Morse L.R., Xu Y., Battaglino R.A., Yang X.F., Jiang Z., Xin H.B, & Fu M. (2015). Adipocyte-derived PAMM suppresses macrophage inflammation by inhibiting MAPK signalling. The Biochemical journal, 472(3), 309-318.

Publication 2015

RNA STAT-60 reagent random priming method

Agarose Buffer Cdna Clone Formaldehyde Gels Hybridization Membranes Mouse Nylon Tissues

Corresponding Organization :

Other organizations : University of South China, University of Missouri–Kansas City, Nanchang University, Harvard University, Temple University

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Variable analysis

independent variables

Total RNA isolation using RNA STAT-60 reagent

dependent variables

PAMM gene expression

control variables

Uniformity of sample loading verified by UV visualization of ethidium bromide-stained gels before transfer to Nylon membranes
Hybridization performed using QuckHyb buffer at 65°C for 2 h or overnight
Membranes washed once with 2 × SSC and once with 0.1 × SSC, 1% SDS for 20 min at 65°C

controls

Positive control: cDNA clone from A.T.C.C. used as templates for PAMM cDNA probe amplification by PCR
Negative control: Not explicitly mentioned

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